Animals live with constant threats to their survival in the form of heat shock, oxidative stress, hypoxia, infection, and toxic challenges from the environment. To combat these threats and maintain homeostasis, animals have evolved complex and specific regulatory responses that include the coordinated transcriptional control of key defensive genes. Many of these regulatory networks have been identified and characterized in the fruit fly Drosophila melanogaster, and shown to be conserved through evolution, providing a foundation for understanding their control in humans. These responses have also provided key insights into the molecular mechanisms of coordinate transcriptional control in higher organisms. Remarkably, one of the most critical defense responses in animals - the ability to detoxify harmful chemicals - has not yet been characterized using a simple genetic model system. These compounds, which include pharmaceuticals, pesticides, plant toxins, and pollutants, referred to collectively as xenobiotics, comprise a critical risk factor for cancer and respiratory diseases. Xenobiotic responses also remain a major impediment to the development of new drugs by the pharmaceutical industry. We have shown that treatment of Drosophila with a widely used xenobiotic, the sedative phenobarbital, results in a rapid, widespread, and coordinate change in gene expression. Many phenobarbital-regulated genes encode enzymes and have proposed functions consistent with detoxification, protection, drug transport, or excretion. A null mutation in DHR96 - the single ancestral fly ortholog of the vertebrate xenobiotic receptors PXR and CAR - affects the phenobarbital regulation of some of these genes, demonstrating that DHR96 can function like its vertebrate counterparts by contributing to xenobiotic responses in insects. Most genes, however, are properly regulated by drug in the absence of DHR96, an effect also seen in mouse PXR and CAR mutants, raising the question of how this coordinate transcriptional circuit is controlled. The goal of this proposal is to use Drosophila to define the molecular mechanisms of xenobiotic transcriptional responses. We will identify critical promoter sequences that are required for phenobarbitalregulated transcription and characterize the trans-acting factors that confer this control. We will also determine the molecular mechanisms by which DHR96 contributes to xenobiotic transcriptional responses. This research will provide new insights into how higher organisms, including humans, cope with toxic compounds in their environment and establish Drosophila as a new model system for characterizing xenobiotic response regulation.